But no matter! I am just as happy helping Laura design her future experiment. Last Tuesday, she gave me a crash course in immunohistochemistry (IHC). I also found a neat video that really clarified the technique for me. Watch it here.
We'll be using indirect IHC to visualize a number of inflammation markers in rodent hippocampal brain slices. But instead of using a marker like DAB (as seen in the video), we'll use fluorescent tags on our secondary antibodies that can be imaged with the confocal microscope.
Right now, we're looking at five different primary antibodies - one for each target molecule - from five different host animals: goat, guinea pig, rat, mouse, and rabbit. So many animals!
I'm also having fun playing with Life Technologies' Fluorescence SpectraViewer to map out which fluorophores we want to use with our secondaries. It is important that each tag's excitation and emission wavelengths are unique to prevent crosstalk and to produce a clear image.
|Good example, adequate spacing.|
|Bad example, overlapping signals.|
Try it out yourself!